G protein linked receptors (GPRs) are a family of integral membrane bound proteins involved in mediating the biological actions of neurotransmitters, hormones and growth actors. They are major targets for pharmaceutical drug development, and a large number of clinically used drugs bind to GPRs. The recent advances of the Human Genome Project have revealed hundreds if not thousands of novel orphan GPRs. These orphan receptors have an almost unlimited potential for new drug development and have generated substantial interest in the pharmaceutical industry for their use as new therapeutic targets. However, discovery of drugs that selectively interact with orphan receptors is hindered by the Act that most GPRs have no known endogenous ligands and therefore can not be detected in ligand binding assays. GPRs couple to G proteins, and agonist stimulation of GPRs promotes the binding of GTP to G proteins. Therefore, measurement of GTP binding to G proteins can be employed as a functional screen for ligand binding to GPRs and orphan receptors. DiscoveRx is a start up biotechnology company that will develop a simple, rapid and sensitive assay for agonist stimulation of the binding of GTPgammaS to G proteins. The assay employs CEDIA technology, which is a homogeneous method for measurement of protein-ligand interactions based on in vitro complementation of enzyme fragments. Formation of active enzyme is regulated by binding of the G protein to a GTPgammaS-enzyme fragment conjugate; the amount of enzyme formed can be determined in solution, without separation or wash steps, using chromogenic or fluorogenic substrates. The goal of this proposal is to develop a novel, easy to use assay for high throughput screening of ligand binding to GPRs. As a proof of principle, DiscoveRx will develop a CEDIA assay to measure the binding of agonists to the cloned mu opioid receptor. In this application, we will optimize the conditions of the CEDIA assay for the detection of opioid stimulation of the mu receptor. These experiments will be the basis of future studies (Phase II application) to determine the general applicability of the DiscoveRx technology to measure drug binding to GPRs and to optimize the assay for high throughput screening of GPRs and orphan receptors. PROPOSED COMMERCIAL APPLICATION: The DiscoveRx assay technology developed in this proposal will be commercialized by providing CEDIA assays (both as kits and as custom assays) and services based on this technology to companies with GPR drug discovery programs, including major pharmaceutical and biotech companies. DiscoveRx will work together with those companies to optimize the assay, much like Aurora Biosciences licenses and optimizes its high throughput screening assays with pharmaceutical companies. The estimated market for these assays will be in the millions of dollars.